The search for a better treatment for recurrent Clostridium difficile disease: use of high-dose vancomycin combined with Saccharomyces boulardii.

Recurrent Clostridium difficile disease (CDD) is a difficult clinical problem because antibiotic therapy often does not prevent further recurrences. In a previous study, the biotherapeutic agent Saccharomyces boulardii was used in combination with standard antibiotics and was found to be effective in reducing subsequent recurrences of CDD. In an effort to further refine a standard regimen, we tested patients receiving a regimen of a standard antibiotic for 10 days and then added either S. boulardii (1 g/day for 28 days) or placebo. A significant decrease in recurrences was observed only in patients treated with high-dose vancomycin (2 g/day) and S. boulardii (16.7%), compared with those who received high-dose vancomycin and placebo (50%; P=.05). No serious adverse reactions were observed in these patients. Comparison of data from this trial with data from previous studies indicates that recurrent CDD may respond to a short course of high-dose vancomycin or to longer courses of low-dose vancomycin when either is combined with S. boulardii.     

Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-α concentration

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-α (TNF-α) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34⁺ compartment from MDS patients (P = 0.039), which are absent in both CD34^? MDS cells (P = 0.53) and also CD34⁺ cells from normal subjects (P = 0.55). MDS CD34⁺ blood cells also showed oxidized pyrimidine nucleotides compared with CD34^? cells (P = 0.029). Within normal subjects no differences were seen between CD34⁺ and CD34^? bone marrow cell compartments. CD34⁺ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-α and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-α, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.     

Altered glycosaminoglycan production by HL-60 cells treated with 4- methylumbelliferyl-beta-D-xyloside

Glycosaminoglycans, mainly chondroitin 4-sulfate, are located in the primary granules of human myeloid cells. These polyanionic carbohydrates are believed to play an important role in leukocyte maturation and function. To study the effect of altered chondroitin sulfate metabolism on human promyelocytic leukemia cells, we have treated HL-60 cells with 4-methylumbelliferyl-beta-D-xyloside. beta-D- Xylosides initiate the synthesis of free chondroitin sulfate chains. Cytochemical studies of treated cells demonstrated a marked increase in cytoplasmic granules stained with cationic dyes. This was confirmed by radiolabeled precursor incorporation studies that demonstrated a 344% increase in 35S-sulfate uptake into glycosaminoglycans associated with the cells and a 39% increase in incorporation into glycosaminoglycans released into the media. Chromatographic analyses of these glycosaminoglycans from treated cells demonstrated that the newly formed chondroitin sulfate chains were not attached to protein core and were of shorter length, but of greater charge density than chondroitin sulfate produced by control cells. Thus, beta-D-xyloside appears to alter the protein linkage, chain length, and sulfation of chondroitin sulfate produced by HL-60 cells, and these changes are morphologically evident. These biochemically altered cells may provide important information concerning the role of these macromolecules in myeloid development.     

Efficacy of commercial condoms in the prevention of hepatitis B virus infection

Tips of synthetic and natural condoms were filled with serum samples containing either hepatitis B virus, herpes simplex virus, or cytomegalovirus, then fit over an 8-in. mechanical vibrator and inserted vibrating into sterile bath solutions for 30 min. Phosphorus 32-labeled hepatitis B and cytomegalovirus molecular probes and viral culture techniques for herpes simplex and cytomegalovirus were used to determine whether leakage of virus had occurred into the surrounding bath solutions. Natural condoms allowed leakage of hepatitis B virus but not herpes simplex virus or cytomegalovirus, whereas synthetic condoms prevented leakage of all viruses. These results suggest that natural condoms might not be effective in preventing sexually transmitted hepatitis B virus infection.     

Peripheral sensory stimulation elicits global slow waves by recruiting somatosensory cortex bilaterally

Slow waves (SWs) are globally propagating, low-frequency (0.5- to 4-Hz) oscillations that are prominent during sleep and anesthesia. SWs are essential to neural plasticity and memory. However, much remains unknown about the mechanisms coordinating SW propagation at the macroscale. To assess SWs in the context of macroscale networks, we recorded cortical activity in awake and ketamine/xylazine-anesthetized mice using widefield optical imaging with fluorescent calcium indicator GCaMP6f. We demonstrate that unilateral somatosensory stimulation evokes bilateral waves that travel across the cortex with state-dependent trajectories. Under anesthesia, we observe that rhythmic stimuli elicit globally resonant, front-to-back propagating SWs. Finally, photothrombotic lesions of S1 show that somatosensory-evoked global SWs depend on bilateral recruitment of homotopic primary somatosensory cortices. Specifically, unilateral lesions of S1 disrupt somatosensory-evoked global SW initiation from either hemisphere, while spontaneous SWs are largely unchanged. These results show that evoked SWs may be triggered by bilateral activation of specific, homotopically connected cortical networks.

Slow waves (SWs) are the predominant cortical rhythm during nonrapid eye movement sleep and anesthesia (1, 2). Since they were first characterized three decades ago, SWs have been linked to a variety of brain functions, including memory consolidation (3–6), homeostatic synaptic plasticity (7), and grouping of other oscillatory events (8–10). Macroscopic recordings (scalp electroencephalogram [EEG]) have revealed that spontaneous global SWs occur approximately once per second (∼1 Hz) and propagate in a stereotypical front-to-back topography through the entire cortex (11). Local electrophysiology has demonstrated that virtually every cortical neuron participates in traveling SWs, exhibiting phase-locked alternation between depolarization (up-state) and hyperpolarization (down-state) (12, 13). During low arousal states, global SWs occur spontaneously but may also be evoked by peripheral sensory stimulation, as well as direct electromagnetic and optogenetic stimulation of the cortex (14–18). However, much remains unknown about the large-scale circuits supporting SW generation and propagation.Here, we investigate the role of the primary somatosensory cortex and its interhemispheric connections in the initiation and propagation of somatosensory-evoked SWs. To this end, we performed widefield optical imaging of cortical dynamics in awake and anesthetized mice expressing fluorescent calcium indicator GCaMP6f in pyramidal neurons. High-resolution mesoscopic imaging across the whole dorsal neocortex allows for a more-precise characterization of globally coherent waves of neural activity than conventional approaches for recording SWs (EEG or local electrophysiology). We demonstrate that unilateral somatosensory stimulation elicits bilateral waves that propagate in opposite directions depending on state (awake versus ketamine/xylazine anesthesia). Further, we show that the imposed rhythm of stimulation induces resonant activity locally in sensorimotor areas that remains focal in awake animals. In contrast, in anesthetized animals, rhythmic stimulation subsequently elicits SWs that spread globally in a front-to-back trajectory across the cortex. Finally, we use a photothrombotic stroke model to show that somatosensory-evoked global SWs depend on engagement of both ipsilateral and contralateral somatosensory cortex (S1). Unilateral photothrombosis of S1 disrupts global SWs evoked by peripheral stimulation of either hemisphere but spares the global spatiotemporal structure of spontaneous SWs outside of the perilesional area. These findings suggest a key role for bilateral recruitment of homotopically connected somatosensory cortices in initiating somatosensory-evoked global SWs and suggest potential mechanisms by which focal cortical injuries may influence global brain dynamics.     

RAS mutation in acute myeloid leukemia is associated with distinct cytogenetic subgroups but does not influence outcome in patients younger than 60 years

The pathogenesis of acute myeloid leukemia (AML) involves the cooperation of mutations promoting proliferation/survival and those impairing differentiation. The RAS pathway has been implicated as a key component of the proliferative drive in AML. We have screened AML patients, predominantly younger than 60 years and treated within 2 clinical trials, for NRAS (n = 1106), KRAS (n = 739), and HRAS (n = 200) hot-spot mutations using denaturing high-performance liquid chromatography or restriction fragment length polymorphism (RFLP) analysis. NRAS mutations were confirmed in 11% of patients (126/1106) and KRAS mutations in 5% (39/739). No HRAS mutations were detected in 200 randomly selected samples. Codons most frequently mutated were N12 (43%), N13 (21%), and K12 (21%). KRAS mutations were relatively overrepresented in French-American-British (FAB) type M4 (P < .001). NRAS mutation was over-represented in the t(3;5)(q21 approximately 25;q31 approximately q35) subgroup (P < .001) and underrepresented in t(15;17)(q22;q21) (P < .001). KRAS mutation was overrepresented in inv(16)(p13q22) (P = .004). Twenty-three percent of KRAS mutations were within the inv(16) subgroup. RAS mutation and FLT3 ITD were rarely coexistent (14/768; P < .001). Median percentage of RAS mutant allele assayed by quantitative RFLP analysis was 28% (N12), 19% (N13), 25% (N61), and 21% (K12). RAS mutation did not influence clinical outcome (overall/disease-free survival, complete remission, relapse rate) either for the entire cohort or within cytogenetic risk groups.     

A field study on the effects of Fort Morgan virus, an arbovirus transmitted by swallow bugs, on the reproductive success of cliff swallows and symbiotic house sparrows in Morgan County, Colorado, 1976

We studied the transmission of Fort Morgan (FM) virus within colonies of nesting Cliff Swallows and House Sparrows under three bridges in Morgan County, Colorado during 1976. Nests were examined, and blood or brain specimens were collected from nestlings once or twice a week. Flying birds and small mammals were also studied. We analyzed nesting activity, virus isolations from nestlings of both species, fledging success, multiple infections within a brood of nestlings, infection frequency by age of nestlings, nestling mortality, and infection frequencies by avian species and bridge site. Fort Morgan virus was isolated from 7% (80/1, 156) of the blood and brain samples collected from nestlings. The duration of viremia for nestling House Sparrows was at least 3-4 days based on virus isolation from sequential blood samples. Viremia of nestling Cliff Swallows and House Sparrows did not reduce fledging success, nor were young nestling sparrows viremic more frequently than older nestling sparrows. Nest destruction (by falling down) was a more important cause of nestling mortality than FM virus infection. All age groups of nestling sparrows were viremic at equal rates, but younger nestlings (less than or equal to 7 days old) were more likely than older nestlings (greater than 7 days old) to develop an encephalitic infection. Among nestling House Sparrows, FM virus infections were clustered in time and space. Nestling House Sparrows with FM virus-infected nest-mates were infected more often than conspecifics whose nest-mates were not infected. We concluded that nestling Cliff Swallows and symbiotic House Sparrows that reside in swallow nesting colonies are the principal vertebrate hosts for the maintenance and amplification of FM virus.     

Family studies in von Willebrand''s disease by analysis of restriction fragment length polymorphisms and an intragenic variable number tandem repeat (VNTR) sequence

We have previously identified a microsatellite variable number tandem repeat region of the nucleotide sequence ATCT within intron 40 of the von Willebrand factor (vWF) gene. By polymerase chain reaction (PCR) amplification of this region, eight major alleles have been demonstrated in the South Wales population, with an overall heterozygosity rate of 75%. Direct sequencing has shown that the alleles correspond to lengths of between six and 14 ATCT repeats. In the present study we describe the use of this variable repeat sequence and previously reported restriction fragment length polymorphisms (RFLP) to study inheritance patterns in families with type I, IIA and severe type III von Willebrand's disease (vWD). The results confirm that analysis of this precisely localized intragenic locus provides a highly informative marker for gene tracking studies in the major forms of vWD.